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VITAL  STAINING  OF  HUMAN  BLOOD  WITH 

SPECIAL  REFERENCE  TO  THE  SEPARATION 

OF  THE  MONOCYTES 


BY 

MIRIAM    E.   SIMPSON 


University  of  California  Publications  in  Anatomy 

Vol.  1,  No.  1,  pp.  1-9 

Issueel  December  6,  1921 


VITAL    STAINING    OF    HUMAN    BLOOD    WITH 

SPECIAL  REFERENCE  TO  THE  SEPARATION 

OF  THE  MONOCYTES* 

BY 

MIRIAM  E.  SIMPSON 
From  the  Anatomical  Laboratory  of  the  University  of  California. 


The  possible  differing  beliavior  of  blood  cells  to  vital  stains  has 
not  yet  been  adequately  explored.  Yet  if  one  will  search  carefully 
the  literature  of  vital  staining  it  will  be  seen  that  Certes,  Mitrophanow, 
Teichmann,  Galeotti,  Ehrlich  and  Muller,  Arnold,  and  Plato,  to  name 
no  others  among  the  earlier  observers,  subjected  the  leucocytes  to  some 
scrutiny  ■  in  experiments  with  vital  dyes.  Furthermore,  during  the 
last  ten  years  two  or  three  significant  attempts  have  been  made  to 
investigate  blood  cells  by  the  supra-vital  application  of  dyes.  I  refer 
especially  to  the  papers  of  Rosin  and  Bibergeil,  Hammar,  Cesaris- 
Demel,  and  Antonio  Ferrata.  This  work  has  made  surprisingly  little 
impression  on  hematology.  It  is  lacking  chiefly,  perhaps,  in  not  being 
essentially  systematic.  Hitherto  no  attempt  has  been  made  to  show 
the  characteristic  behavior  of  living  blood  cells  toward  dyes  repre- 
sentative of  the  different  dye  groups,  nor  has  there  been  sufficient 
study  of  the  differential  behavior  of  the  various  types  of  blood  cells 
toward  any  one  dye  substance.  We  are  not  without  justification, 
however,  in  hoping  that  such  studies  will  throw  light  on  the  physiology 
of  the  leucocytes  and  above  all  on  interrelationships  between  the  vari- 
ous white  blood  cells.  These  studies,  begun  in  the  Anatomical  Labor- 
atory of  the  University  of  California  in  1917  by  M.  C.  Silverberg  and 
A.  C.  Silbermann,  were  carried  further  in  the  next  succeeding  years 
by  Robert  T.  Trotter.  At  the  suggestion  of  Professor  Evans  and  with 
his  aid,  the  present  writer  has  within  the  last  two  years  attempted  to 
make  a  comprehensive  study  of  the  behavior  of  living  human  blood 


*  This  paper  and  the  following  one  are  preliminary  accounts  of  work  accom- 
plished during  the  last  two  years  and  described  in  full  in  theses  formally  filed 
with  the  Dean  of  the  Graduate  School  of  the  University  of  California  on  Sep- 
tember 6,  1921.    The  complete  illustrated  publications  will  appear  elsewhere. 


2  University  of  California  Publications  in  Anatomy        [Vol.  i 

cells  whou  subjected  iu  tliiu  films  to  the  action  of  representative  or- 
ganic dyestuffs.  The  paper  is  one  of  a  series  on  the  reaction  of  the 
tissues  under  normal  and  pathological  conditions  studied  by  means 
of  vital  stains.  About  one  hundred  and  fifty  dyes  of  known  chemical 
constitution  were  compared  in  respect  to  their  action  on  living  leuco- 
cytes.   The  dyes  were  representative  of  all  of  the  dye  families. 

The  method  employed  was  one  which  has  been  ascribed  to  Rosin 
and  Bibergeil  (1902-04),  but  in  its  use  Pappenheim  disputes  the 
priority.^ 

The  end  of  a  slide  which  has  been  dipped  in  an  alcoholic  solution 
of  the  dye  is  drawn  across  the  warm  surface  of  another  slide.  This 
leaves  a  thin  film  of  solid  dye  on  the  second  slide,  a  film  consisting  of 
very  minute  particles  which  will  redissolve  rapidly.  A  small  drop  of 
blood  is  then  placed  on  a  cover  slip,  and  the  cover  slip  allowed  to  come 
in  contact  with  the  slide.  If  the  glassware  is  scrupulously  clean  and 
possesses  an  even  surface,  the  blood  will  spread  evenly  and  quickly 
to  the  margin,  or  better,  almost  to  the  margin  of  the  cover  slip.  The 
cover  slip  is  then  quickly  rimmed  with  paraffin  or  lanolin.  The  Avhole 
procedure  must  be  carried  through  in  a  few  seconds  after  obtaining 
the  fresh  drop  of  freely  flowing  blood.  For  making  the  dye  "films," 
solutions  of  varying  concentration  should  be  employed.  A  1  per  cent 
alcoholic  solution  of  the  dye  was  the  concentration  systematically  tried 
first.  Then,  judging  by  the  results  obtained,  lower  and  lower  concen- 
trations were  employed.  At  times  even  a  .001  per  cent  solution  gave 
results.  Such  beautiful  preparations  supply  one  with  a  film  consist- 
ing of  a  single  layer  of  living  blood  cells  spread  out  in  the  most  ad- 
vantageous position  for  study.  With  experience  injury  to  the  cells 
from  handling  is  minimal,  and  in  totally  unstained  preparations  or  in 
those  made  with  very  weak  solutions  of  many  dyes,  living  healthy  cells 
may  be  observed  pushing  out  pseudopodia  or  moving  in  amoeboid 
fashion  for  several  hours.  Illumination  through  Gage 's  daylight  glass 
and  apochromatic  oil  lenses  were  employed. 

Dyes  having  the  greatest  differences  in  chemical  and  physical 
properties  may  enter  and  may  be  stored  by  the  living  blood  cells.  As 
regards  chemical  make-up  of  the  dyes,  the  following  table  will  give 
the  number  of  representatives  of  the  various  dye  groups  which  were 
employed,  and  their  positive  or  negative  behavior  as  supra-vital  stains. 


1  For  further  discussion  of  the  priority  in  the  use  of  this  method  see  the 
discussion  by  Hammar  (1912)  and  by  Pappenheim  (1907). 


1921]  Simpson:  Vital  Staining  of  Human  Blood  3 

Dye  group  Positive*        Negative* 

Nitroso  1 

Nitro  1 

Stilbene  2  1 

Pyrazoleu 1 

Monazo 6 

Disazo  3  4 

Triphenylmethane    2  17 

Xanthones    6  7 

Acridines   1  1 

Oxazine 13  8 

Thiazine  5  5 

Azine — 

(b)  Eurhodine  1 

(c)  Aposafranine  2 

(d)  Benzosafranine 19  5 

2.  Naphtosafranine   1  2 

(e)  Induline 1  2 

Anthraquinone    2  10 

Indigo  2  1 

Inorganic   2 

Totals   81  73 

*  "Positive"  means  that  the  dye  was  observed  in  the  cytoplasm  of  cells  either  diffusely,  in  the 
specific  granules,  in  mitoehondria  or  in  special  storage  granules,  before  the  nucleus  stained. 
"Negative"  means  either  that  the  dye  was  not  seen  to  penetrate  the  cell  at  all,  or  that  if  it  did 
appear  in  the  cell,  death  of  the  cell  was  coincident  with  the  entrance  of  the  dye. 

The  supra-vital  dyes  may  stain  structures  which  existed  in  the 
cell  at  the  time  of  treatment  with  the  dye  and  which  are  therefore 
comparable  to  structures  stained  in  fixed  preparations.  One  may 
therefore  obtain  satisfactory  vital  stains  of  the  specific  granules  of  the 
polymorphonuclear  leucocytes.  Supra-vital  methods  may  indeed  pro- 
duce the  most  striking  stains  of  the  specific  granules.  I  may  enumer- 
ate here  Victoriablau  B,  Victoriablau  4R,  Neumethyleneblau  N,  Neu- 
methyleneblau  GG,  Neuechtblau  F,  Nile  Blue  R,  Nile  Blue  A,  Nile 
Blue  B  extra,  Nile  Blue  BB,  Methylene  Blue  Med.,  Capri  Blue  GON, 
Brilliant  Cresyl  Blue,  Thionin  Blue  GO,  Toluidin  Blue,  Anthrachi- 
nongrun  GXNO,  Brilliantrhodulin  Red  B,  Indazine  M. 

Very  few  instances,  however,  were  found  of  dyestuffs  which  stained 
these  structures  alone,  and  in  those  instances  the  dyes  emploj^ed  could 
not  be  called  good  vital  stains.  The  above  mentioned  dyes,  besides 
staining  specific  granules,  may  stain  beautifully  the  "segregation" 
granules,  to  be  mentioned  later. 

As  Laguesse,  Michaelis,  Bensley,  and  Cowdry  have  shown,  mito- 
chondria may  be  electively  stained  by  vital  dyes.  The  best  example 
of  such  a  dye  is  Janus  Green  B,  to  which  it  is  now  possible  to  add 
Amethyst  Violet,  Iris  Violet,  Janus  Grey  2B,  Janus  Dark  Blue  R, 
Diazine  Black,  Janus  Black  II,  Naphtindon,  Echtneutralviolett. 


4  University  of  CaUfornia  PuhUcaiions  in  Anatomij        [Vol.  l 

The  mitochondrial  content  of  the  mononuclear  cells  is  higher  than 
that  of  the  polyniorphonuelear  cells.  Among  the  polymorphonucltar 
cells  the  mitoeliondria  are  more  numerous  and  easier  to  demonstrate 
in  the  neutrophils  than  in  the  eosinophils.  The  basophils  are  too  in- 
frequent in  human  blood  to  be  placed  readily  in  such  a  series.  From 
work  on  the  rabbit,  however,  where  basophils  are  abundant,  the  mito- 
chondrial content  of  the  basophils  has  been  shown  to  be  very  low.  In 
the  mononuclear  cells  tlie  number  of  mitochondria  present  seems  to 
be  a  function  of  the  amount  (size)  of  the  cytoplasm,  being  invariably 
numerous  in  the  transitionals  and  always  more  numerous  in  the  larger 
than  in  the  small  lymphocytes. 

The  rieli  mitochondrial  content  both  of  large  lymphocytes  and  of 
monocytes  makes  it  impossible  by  this  means  to  discriminate  between 
the  lymphocytes  and  the  other  important  group  of  mononuclear  cells 
to  which  the  name  monocyte  has  now  been  given  (Naegeli).  Fortu- 
nately, in  the  case  of  other  vitally  stained  granules — the  segregation 
granules — such  a  distinction  between  lymphocytes  and  monocytes  is 
now  possible,  as  will  be  described  later. 

The  lymphocytes  are  particularly  well  adapted  for  the  observation 
of  mitochondria  in  fresh  blood  preparations,  not  only  because  they 
are  numerous  in  these  cells  but  because  of  the  almost  complete  absence 
of  other  granules.  Only  an  occasional  refractive  vacuole  and  two  or 
three  "segregation  granules"  are  present. 

Cowdry  (1914,  1916)  has  spoken  of  the  specificity  of  the  staining 
of  mitochondrial  substance  to  the  safranine  chemical  nucleus  and  also 
of  the  importance  of  the  substituted  groups.  In  this  study  the  safra- 
nine derivatives  were  found  to  include  all  the  best  mitochondrial 
stains.  However,  dyes  representative  of  at  least  three  Schultz  groups 
were  found  to  contain  mitochondrial  stains.  It  is  therefore  evident 
that  the  safranine  nucleus  can  not  be  regarded  as  a  specific  require- 
ment for  mitochondrial  stains.  The  importance  which  has  been  placed 
on  the  ethyl  group  in  mitochondrial  stains  has  also  probably  been  over- 
estimated. This  seems  to  be  true  even  in  the  azine  group  (safranine 
nucleus)  where  the  ethyl  group  has  been  considered  by  Cowdry  (1916") 
to  be  the  factor  which  allowed  Janus  Green  B  to  stain  mitochondria 
while  Janus  Green  G,  of  the  same  constitution  except  in  this  substi- 
tution of  the  two  ethyl  groups  of  Janus  Green  B  by  methyl  groups, 
was  not  a  mitochondrial  stain.  Naphtindon  (Safranine-Beta  Napli- 
thol)  is  an  example  of  a  mitochondrial  stain  which  contains  neither 
ethyl  nor  methyl  groups.  In  other  dye  classes  containing  mitochon- 
drial stains,  some  of  the  dyes  contain  only  methyl  groups  (two,  four. 


1921]  Simpson:  Vital  Staining  of  Human  Blood  5 

or  six)  substituted  for  the  hydrogen  of  the  amine  groups,  others 
contain  only  ethyl  groups,  and  still  others  contain  both  groups.  It 
would  therefore  seem  that  these  groups  are  not  specific  for  the  mito- 
chondrial reaction. 

Many  dyes  are  attracted  to  and  concentrated  in  a  special  set  of 
granules  and  vacuoles  in  certain  cells,  structures  to  which  Evans  and 
Scott  have  given  the  term  "segregation  apparatus,"  and  which  are 
largely,  although  not  exclusively,  preformed  structures.  These  struc- 
tures are  present  in  blood  cells.  They  may  be  brilliantly  displayed 
by  vital  stains.  The  vacuoles  often  have  the  power  to  increase  in  size 
as  the  amount  of  the  dye  stored  in  them  increases.  This  reaction  on 
the  part  of  the  living  cell  may  be  regarded  as  a  special  contrivance 
to  isolate  from  the  protoplasm  and  hence  render  harmless  either  ex- 
cretory products  or  foreign  materials  forced  on  the  cell.  The  reaction 
is  given  best  perhaps  by  Neutral  Red,  Nile  Blue  Sulfate,  and  Brilliant 
Cresyl  Blue,  but  over  a  score  of  other  dyes  were  found  to  behave 
typically  in  this  way. 

Ferrata,  Rosin  and  Bibergeil,  Dubreuil,  Cesaris-Demel,  and  Ham- 
mar  have  especially  studied  the  granules  which  may  be  produced  in 
blood  cells  by  the  application  of  this  group  of  dyes.  In  order  to 
justify  a  term  like  "segregation  apparatus,"  it  is  necessary  to  sep- 
arate these  granules  from  the  specific  granules  and  from  the  mito- 
chondria ;  this  distinction  can  be  made.  Both  the  size,  shape,  and 
number  of  the  granules  are  good  criteria  for  the  differentiation  of  the 
segregation  apparatus  from  mitochondria.  With  dyes  like  Janus 
Green  B  and  Neutral  Red  the  granular,  rod,  and  filamentous  forms 
of  the  mitochondria  are  especially  contrasted  with  the  globules  of  the 
segregation  apparatus  because  of  the  peculiar  tendency  of  the  latter 
structures  to  increase  rapidly  in  size.  Yet  it  would  not  be  justifiable 
to  assume  that  all  globular  structures  are  segregation  bodies  and  all 
filamentous  forms  mitochondria.  The  best  mitochondrial  stains,  in- 
cluding Janus  Green  B,  eventually  distort  the  mitochondrial  morphol- 
ogy, and  they  may  do  this  very  rapidly  if  high  concentrations  are 
employed.  In  the  neutrophils,  eosinophiles,  and  monocytes  the  differ- 
ence in  the  number  of  granules  in  the  segregation  apparatus  and  the 
number  in  the  mitochondrial  apparatus  is  not  a  conspicuous  feature. 
It  is  in  the  lymphocytes  that  there  is  a  marked  difference  in  the  num- 
ber of  the  two  sets  of  granules.  Double  staining,  for  example,  with 
Neutral  Red  and  Janus  Green  M^ell  illustrates  this  difference.  In  the 
case  of  the  lymphocytes,  the  contrast  between  the  few  red  granules  of 


6  University  of  CcUifarnia  Puhlications  in  Anatomy        [Vol.  l 

the  segregation  apparatus  (from  one  to  three  or  four)  and  the  numer- 
ous mitochondria  (from  ten  to  twenty  or  more)  is  always  demon- 
strable. 

The  low  content  of  lymphocytes  in  segregation  granules  and  the 
exceptionally  high  content  of  monocytes  enables  ns  to  separate  these 
cells.  The  classification  of  the  mononuclear  cells  of  the  blood  has 
been  a  source  of  dispute  among  hematologists  for  years.  Agreement 
was  easily  reached  on  the  lymphatic  origin  of  the  small  cells.  It  was 
not  so  simple  a  matter,  however,  to  decide  on  the  origin  and  relation- 
ships of  the  larger  mononuclear  cells.  The  early  classifications  of  the 
mononuclear  cells  were  based  on  morphology  and  fixed  staining  re- 
actions and  led  to  widely  different  ideas  of  the  relationships  of  these 
cells.  Ehrlich  separated  them  into  lymphocytes,  large  mononuclears, 
and  transitionals.  The  latter  two  groups  represented,  according  to 
Elirlieh,  different  stages  of  conversion  from  lymphocj^tic  to  the  poly- 
morphonuclear cells.  Weidenreich  saw  among  the  mononuclears  only 
one  type  of  cell,  the  lymphocyte.  The  introduction  of  biological  cri- 
teria has  not  lessened  the  complexity  of  the  subject.  Naegeli  (1919), 
on  the  basis  of  morphological  staining  and  biological  reactions,  divides 
the  mononuclears  into  two  classes,  the  lymphocytes  and  the  monocytes 
(including  the  large  mononuclears  and  transitionals  of  Ehrlich). 
Monocj^tes  are  by  him  supposed  to  constitute  an  independent  group 
of  cells  of  myeloid  origin.  His  morphological  criteria  for  recognizing 
these  cells  consist  in  their  possession  of  a  characteristic,  abundant, 
fine,  dustlike  granulation  stained  by  Azur  and  the  fact  that  these  cells 
give  a  positive  oxydase  response.  Biological  affiliations  are  shown 
by  cases  where  stimulation  of  myeloid  cells  also  leads  to  stimulation 
of  the  monocytes,  and  cases  where  inhibition  of  these  cells  also  inhibits 
the  monocytes,  while  stimulation  of  lymphatic  tissue  does  not  influence 
the  monocyte  count.  McJunkin  (1919)  has  also  applied  morpholog- 
ical, staining,  and  biological  methods  and  draws  widely  different  con- 
clusions; but  although  McJunkin,  with  Mallory  and  others,  classifies 
these  questionable  large  mononuclears  of  the  normal  blood  as  deriva- 
tives of  endothelium  and  uses  the  term  "endothelial  leucocytes"  to 
designate  them,  sufficient  evidence  to  justify  this  term  has  not  been 
produced.  In  some  cases  he  used  a  modified  oxydase  method  for  recog- 
nizing the  cells.^    McJunkin 's  biological  test  consists  in  determining 

-  The  application  of  the  oxydase  as  a  method  for  aeparating  those  colls  in  some 
mammals  has  proved  exceedingly  disappointing.  Equally  capable  hematologists 
report  a  negative  as  well  as  a  positive  response  on  the  part  of  the  transitionals 
or  monocyte  cell. 


1921]  Simpson:  Vital  Stadning  of  Human  Blood  7 

phagocytic  function  under  a  definite  set  of  conditions.  He  finds  one 
set  of  conditions  under  which  the  vascular  endothelium  and  these  large 
mononuclear  cells  of  the  blood  are  the  only  cells  which  phagocytize 
carbon,  following  intravenous  carbon  injections,  and  he  deduces  that 
the  transitions  are  derivatives  of  this  endothelium.  Furthermore, 
while  there  could  be  no  doubt  that  the  transitional  leucocytes  are  very 
much  more  active  phagocytes  than  are  the  lymphocytes,  and  in  these 
respects  are  like  the  specific  endothelia,  it  is  unlikely  that  one  can 
produce  conditions  which  will  force  every  transitional  to  exhibit  this 
activity.  At  any  rate,  in  these  particular  experiments  McJunkin  used 
no  other  criteria  save  carbon  injection  to  recognize  the  transitional 
cells,  and  admits  that  a  certain  proportion  of  these  cells  did  not  con- 
tain the  carbon. 

Ferrata  might  also  be  considered  to  have  applied  biological  criteria 
when  he  used  the  behavior  of  the  mononuclear  cells  to  supra-vital 
stains  as  a  method  of  classification.  But  Ferrata  (1908)  believed  that 
the  supra-vital  staining  furnished  strong  evidence  for  the  derivation 
of  all  the  mononuclear  cells  from  lymphatic  tissue,  and  considered  that 
morphological  differences  were  due  to  ageing  of  the  cells.  Ferrata 's 
evidence  may  be  summarized  as  follows : 

(1)  The  cells  form  a  complete  series;   all  intermediate  sizes  are  present  in 

the  blood. 

(2)  As  the  cells  become  larger  the  nucleus  stains  less  intensely. 

(.3)   Metachromatic  droplets  (Brilliant  Cresyl  Blue)  appear  only  in  the  larger, 
older  cells. 

(4)  The  collection  of  Brilliant  Cresyl  Blue  in   "plasmasomes"  is  common 

to  all  members  of  this  group  and  distinguishes  these  cells  from  the 
polymorphous  cells. 

(5)  Fat  droplets  occur  in  all  the  mononuclear  cells. 

(6)  The  nuclei  have  similar  proportions  in  all  the  mononuclear  cells. 

Ferrata  placed  his  confidence  in  the  existence  of  but  one  type  of 
mononuclear  cell  in  the  blood  on  the  fact  that  all  intermediate  forms 
would  be  seen  among  the  living,  supra-vitally  stained  cells.  It  was 
therefore  of  the  greatest  interest  to  discover  that  the  supra-vital  method 
furnishes  one  of  the  most  valuable  methods  of  distinguishing  two 
groups  of  mononuclear  cells  in  the  blood.  The  method  has  not  only 
been  shown  to  be  a  satisfactory  one  for  the  study  of  human  blood  but 
has  been  applied  to  other  mammals,  e.g.,  rabbits,  and  has  been  found 
of  far  greater  value  for  the  identification  of  transitionals  in  experi- 
mental blood  studies  in  that  animal  than  either  Giemsa  or  oxydase 
stains. 


8  Utiivcrdti/  of  California  Fuhlications  in  Anatomy        [Vol.1 

Ferrata  is  correct  in  so  far  as  all  intermediate  sizes  of  lymphocytes 
from  small  to  large  can  be  found.  However,  there  are  very  few  of 
the  lymphoeyte-s  which  reach  a  size  which  is  as  large  as  the  average 
size  of  monocytes.^  Yet  most  of  the  characteristics  of  these  two  kinds 
of  mononuclear  cells  were  found  to  be  shared  in  common,  though  these 
characteristics  were  expressed  in  various  degrees.  A  few  character- 
istics were  observed,  however,  which  were  definitely  not  held  in  com- 
mon and  which  served  to  separate  two  types  of  cells.  The  cytoplasm 
of  the  lymphocytes,  both  in  the  smallest  and  in  the  largest  forms,  had 
a  clear  hyalin  nature,  so  that  the  cell  appeared  distinctly  different 
from  the  transitional  cells  with  their  ground  glass  or  finely  granular 
cytoplasm.  This  criterion  was  of  minor  importance  compared  with 
the  differences  which  were  found  to  exist  in  the  segregation  apparatus. 
The  segregation  apparatus  did  not  furnish  an  absolute  point  of  sep- 
aration between  polymorphonuclear  and  mononuclear  cells  as  had  been 
supposed  by  Ferrata.  This  set  of  granules  is  present  in  both  cell 
types.  Qualitatively  the  segregation  apparatus  was  alike  in  all  of 
the  mononuclear  cells,  that  is,  the  rate  of  accumulation  and  final  size 
reached  by  the  structures  was  practically  the  same  in  all  the  mono- 
nuclear cells.  The  nuniber  of  granules  characteristic  of  the  segrega- 
tion apparatus  of  the  two  cell  types  is  very  different.  In  the  lymph- 
ocytes there  were  one  to  eight  granules,  depending  on  the  size  of  the 
cell.  On  the  average  the  segregation  apparatus  of  the  lymphocytes 
consisted  of  two  to  three  granules.  In  the  transitional  cells  these 
granules  are  always  numerous.  Forty  to  sixty  granules  would  be  a 
reasonable  estimate  of  the  number  always  found  in  this  cell  type. 
Since  the  granules  can  be  seen  in  fresh  unaltered  living  cells  without 
the  use  of  dyes,  and  since  they  are  especially  clear  in  dark  field  ob- 
servations, these  criteria  can  be  applied  to  separate  even  unstained 
living  mononuclear  cells.*  These  differences  are  rendered  strikingly 
clear  by  dyes  which  accentuate  the  segregation  apparatus  such  as 
Neutral  Red,  Brilliant  Cresyl  Blue,  and  Nile  Blue  Sulfate.     These 


3  The  term  "monocyte"  is  used  in  this  paper  to  incluile  both  the  transitionals 
and  large  mononuclears  of  Ehrlich.  This  distinction  made  by  Ehrlich  was  based 
on  nuclear  indentation.  Heniatologists  are  coming  to  lay  less  and  less  stress  on 
nuclear  form  in  separating  the  mononuclear  cells,  and  in  living  cells  it  is  a  dis- 
tinctly unreliable  distinction.  The  living  nucleus  is  continually  changing  shape. 
The  nuclear  form  of  these  large  mononuclear  cells  is  particularly  motile. 

*  Naegeli  clearly  recognized  the  fact  that  his  so-called  specific  granules  of 
monocytes  were  not  artifacts,  for  they  could  be  seen  in  the  living  cell.  It  is  all 
the  more  remarkable  that  he  was  not  sufficiently  impressed  with  this  to  urge 
that  the  distinction  between  these  cells  and  the  lymphocytes  could  thus  be  ade- 
quately seen  in  the  living  unaltered  cell.  His  method  for  the  recognition  of  these 
cells  consisted  in  the  azurophilic  reaction  of  the  fine  monocyte  granules  in  a  good 
Giemsa  stain. 


1921]  Simpson:  Vital  Stcdmng  of  Human  Blood  9 

facts  were  not,  consequently,  overlooked  by  Hammar  in  his  studies 
with  Brilliant  Cresyl  Blue,  though  he  looked  upon  the  segregation 
structures  as  degenerative  products  produced  by  autolysis.  It  is  of 
distinct  interest  that  the  vital  stains  enable  us  to  deny  Naegeli's  con- 
ception of  the  peculiar  or  specific  nature  of  the  granules  of  the  transi- 
tionals  or  monocytes,  for  they  show  that  the  granules  of  monocytes 
are*  at  any  rate  essentially  the  same  in  nature  as  those  possessed  by  all 
blood  cells.  They  consist  of  mitochondria  and  of  segregation  granules 
and  merely  the  number  of  the  latter  is  very  significantly  increased.^ 

The  supra-vital  application  of  certain  dyes  to  human  blood  has  been 
shown  to  furnish  an  excellent  method  for  distinguishing  the  special 
group  of  mononuclear  cells  comprised  originally  of  the  transitionals 
and  large  mononuclears  of  Ehrlich — the  monocytes.  It  is  of  interest 
that  these  cells  and  they  alone  stand  in  some  sort  of  relation — as  yet 
not  wholly  clear — to  the  endothelial  macrophages  which  may  be  ex- 
perimentally produced  in  the  mammalian  body  by  a  variety  of  pro- 
cedures which  will  be  summarized  in  a  later  paper. 


5  The  identity  or  non-identity  of  the  vitally  stained  granules  with  the  sporad- 
ically occurring,  so-called,  azurophilic  granulation  of  lymphocytes  discovered  by 
Michaelis  and  Wolff  (1902)  has  interested  several  observers.  Hammar  treated 
a  preparation  of  vitally  stained  cells  with  a  double  May-Grunwald  and  Giemsa 
stain  (after  Pappenheim).  He  decided  that  neither  in  position,  size,  or  number 
were  the  two  kinds  of  granules  the  same.  Betances  (1918)  reached  a  similar 
conclusion  although  Ferrata  concluded  they  were  the  same  structures.  Further- 
more, most  lymphocytes  normally  show  a  few  segregation  bodies,  but  at  best 
only  a  portion  of  them  possess  the  azurophilic  granules.  Naegeli  argues  con- 
vincingly that  the  monocytic  granules  are  never  brilliant  red  as  are  the  lympho- 
cytic ones,  that  they  are  smaller,  and  are  invariably,  not  occasionally,  present. 


Transmitted  September  6,  1921. 
(See  footnote,  page  1.) 


THE  EXPERIMENTAL  PRODUCTION  OF  CIR- 
CULATING  ENDOTHELIAL  MACROPHAGES 
AND   THE   RELATION   OF  THESE   CELLS   TO 
THE  MONOCYTES 

BY 

MIRIAM   E.   SIMPSON 


University  of  California  Publications  in  Anatomy 
Vol.  1,  No.  2,  pp.  11-19 
Issued  December  6,  1921 


THE  EXPERIMENTAL  PRODUCTION  OF  CIRCU- 
LATING   ENDOTHELIAL    MACROPHAGES 
AND   THE   RELATION   OF   THESE 
CELLS  TO  THE  MONOCYTES 

BY 

MIEIAM  E.  SIMPSON 

From  the  Anatomical  Laboratory  of  the  University  of  California. 


The  great  significance  of  certain  large  phagocytic  cells  in  the 
metabolic  and  protective  reactions  of  the  body  is  just  beginning  to 
be  known.  These  cells  have  been  variously  called  pyrrhol  cells  (Gold- 
man), adventitia  cells  (Marchand),  rhagiocrine  cells  (Renaut),  rest- 
ing wandering  cells  (Maximow),  endothelial  leucocytes  (Mallory), 
clasmatocytes  (Ranvier),  histiocytes  (Aschoff,  Kiyono),  macrophages 
(Evans),  Kupfer  cells  and  the  other  "specific  endothelia"  in  the 
lymph  glands,  hemal  nodes,  bone  marrow,  and  spleen.  In  the  study 
of  these  cells  endless  discussion  has  arisen  on  their  relationship  to 
other  cells  and  tissues  of  the  body  and  on  the  relationships  between 
these  cells  themselves.  Due  largely  to  recent  -work  with  vital  stains, 
done  first  by  Bouffard  but  really  inaugurated  with  greater  care  by 
E.  E.  Goldman  and  continued  by  Kiyono  and  by  Evans  and  Schule- 
mann,  we  now  know  that  these  cells  have  certain  common  character- 
istics, chief  of  which  is  the  power  to  receive,  store,  and  concentrate 
within  their  protoplasm  a  large  group  of  coloring  matters  belonging 
to  the  acid  azo  dyes.  Correlative  with  this  behavior  is  their  activity 
as  phagocytes.  The  idea  of  classifying  these  apparently  diverse  ele- 
ments in  one  cell  group  was  first  suggested  by  H.  M.  Evans  (1915), 
who  reemphasized  the  early,  fundamental  immunological  work  of 
Metchnikoff  in  resurrecting  the  term  "macrophage"  for  the  cells  of 
this  group.  In  particular,  it  would  seem  unfortunate  to  adopt  the  term 
"  clasmatocyte "  for  the  connective  tissue  members  of  this  cell  group, 
for  Ranvier 's  term,  as  Maximow  has  shown,  was  based  on  two  errors: 
first,  his  identification  of  the  mast  cells  of  Amphibia  with  the  connec- 
tive tissue  macrophages  of  Mammals;  and,  second,  the  notion  that 
these  cells  undergo  what  he  called  "  clasmatosis, "  i.e.,  a  pinching  off 
or  abstriction  of  portions  of  their  protoplasm  and  solution  of  the  same 
in  the  tissue  juices — a  process  which  does  not  occur. 


12  University  of  California  Puhlications  in  Anatomy        ["^^ol.  i 

The  cells  would  ordinarilj^  be  classified  as  belonging  to  the  connec- 
tive tissue,  to  the  endothelium  or,  in  rarer  instances,  to  the  blood  stream. 
As  Goldmann  first  showed  and  as  has  been  more  fully  demonstrated  by 
the  work  of  Evans  and  Schulemann  and  of  Evans  and  Scott,  they  con- 
stitute one  of  the  two  great  groups  of  connective  tissue  cells.  Further- 
more, the  cells  line  the  capillaries  and  the  sinusoids  in  the  liver,  lymph 
glands,  hemal  nodes,  bone  marrow,  and  spleen.  It  is  of  great  interest, 
moreover,  that,  in  animals  subjected  to  chronic  treatment  with  lithium 
carmine  or  benzidine  dyes,  the  capillaries  or  sinusoids  in  these  five 
localities  do  not  merely  have  their  endothelial  walls  densely  loaded 
with  the  injected  substance  but  also  contain  large,  free,  similarly 
marked  cells.  These  cells  have  been  especially  studied  by  Evans  and 
his  collaborators  and  by  Kiyono  and  Aschoff.  Entirely  independently 
of  this  work  and  dating  back  for  a  considerable  number  of  years, 
pathologists  have  recognized  that  in  a  variety  of  diseases  large  phago- 
cytic cells  are  developed ;  and  that  such  cells  are  not  merely  intersti- 
tially  placed  but  may  lie  in  blood  vessels.  F.  B.  Mallory,  who  early 
recognized  these  facts,  has  termed  these  cells  endothelial  leucocytes; 
Aschoff  has  designated  them  histiocytes,  and  Evans  has  spoken  of  them 
as  endothelial  macrophages  which  have  become  free  to  join  the  blood 
current. 

The  extent  to  which  these  large  mononuclear  phagocytic  cells  are 
found  in  the  actual  circulating  blood  is  not  as  yet  known  to  us ;  for, 
being  relatively  rare  and  atypical,  they  have  undoubtedly  not  been 
recognized  or  catalogued  with  any  frequency.  But  a  rapidly  increas- 
ing number  of  reports  of  these  cells  has  been  appearing  in  the  litera- 
ture, and  we  now  know  that  a  considerable  number  of  pathological 
conditions  are  included  in  the  cases  showing  this  response.  Ehrlich 
very  early  reported  the  presence  of  large  phagocytic  mononuclears 
in  the  case  of  paroxysmal  haemoglohinuria.  Schilling  has  reported 
macrophages  in  several  other  conditions.  He  has  made  his  most  care- 
ful study  of  the  cells  as  they  appeared  in  ulcerative  endocarditis 
(1919).  A  case  of  Lihm,an's  suhacute  hacterial  endocarditis  has  been 
followed  in  connection  with  this  study  and  the  cell  phenomena  observed 
will  be  discussed  later  in  connection  with  their  bearing  on  the  results 
of  the  experimental  work. 

The  presence  of  these  cells  in  the  blood  stream  of  animals  which 
had  been  injected  chronically  with  various  vital  dyes,  while  denied  by 
the  early  workers  (e.g.,  Goldmann),  was  noticed  by  Schulemann,  who, 
however,  barely  mentioned  the  fact  of  their  occasional  occurrence. 


1921]  Simpson:  Endothelial  Macrophages  13 

Evans  and  Winternitz  in  1911  again  had  occasion  to  notice  frequently 
the  presence  of  these  strange,  large,  brilliantly  colored  cells  in  the 
blood  stream  in  rabbits  which  had  been  injected  chronically  with 
Trypan  Blue  for  studies  on  vital  staining  and  milliary  tuberculosis. 
They  picked  up  these  cells  in  the  blood  from  the  ear  vein.  The  first 
careful  reports  on  the  presence  of  these  cells  in  the  blood,  however, 
was  given  by  Aschoff  and  Kiyono  (1913)  and  by  Kiyono  (1913-14). 
They  found  the  cells  in  the  circulation  after  repeated  intravenous 
injections  of  lithium  carmine  and  established  the  fact  that  the  cells 
were  relatively  rare  throughout  most  of  the  blood  stream  but  relatively 
frequent  in  the  veins  draining  the  five  organs  mentioned  above. 

From  the  above  account  it  is  clear  that  work  from  a  number  of 
different  sources — above  all  the  histological  observations  of  Mallory 
and  the  work  with  vital  carmine  or  benzidine  stains  with  which  Kiyono 
and  Evans  may  be  identified — has  ^11  conspired  to  show  clearly  that  the 
endothelium  in  certain  particular  localities  is  especially  active,  creat- 
ing cellular  products  which  are  probably  of  great  importance  in  the 
bodily  mechanism. 

Besides  the  interesting  questions  Avhich  must  now  be  raised  regard- 
ing the  specific  function  of  these  cells,  the  question  of  their  relation 
to  the  other  white  cells  is  an  important  one.  It  is  possible  that  certain 
of  the  normal  leucocytes  are  endothelial  derivatives  and  hence  also 
to  be  termed  "macrophages,"  although  at  a  lower  stage  of  develop- 
ment than  the  great  cells  met  with  in  disease  or  after  the  use  of  vital 
stains.  Patella  (1909),  for  instance,  as  is  well  known,  though  with- 
out important  confirmation,  has  urged  the  derivation  of  all  the  mono- 
nuclear leucocytes  from  endothelium.  Aschoff  and  Kiyono  jumped 
to  the  conclusion  that  the  large,  carmine-laden  cells  which  they  found 
in  the  blood  stream  in  the  localities  mentioned,  in  addition  to  their 
endothelial  origin,  were  also  to  be  identified  with  the  transitional 
leucocytes  of  Ehrlich.  They  do  not  discuss  the  fact  that  the  more 
normal  sized  and  abundant  transitionals  throughout  the  circula- 
tion were  not  so  stained,  nor  do  they  present  any  detailed  evidence 
justifying  their  surmise. 

Mallory  and  McJunkin  have  not  hesitated  to  identify  the  group 
of  large  mononuclear,  or  transitional  leucocytes  of  Ehrlich  with  the 
unusual,  large  cells — the  true  macrophages — and  consequently  desig- 
nate transitional  leucocytes  as  endothelial  leucocytes.  Their  criteria 
for  this  venture,  however,  have  never  been  adequate  and  this  fact  has 
naturally  led  to  reserve  on  the  part  of  other  students.    At  one  time 


14  University  of  California  Publications  in  Anatomy        l^oh.  i 

McJuiikin  fancied  tliat  he  could,  so  to  speak,  mark  out  these  cells  in 
experimental  animals  by  the  physiological  test  of  phagoeytized  intra- 
venously injected  carbon;  at  another  time  he  proposed  the  oxydase 
reaction  as  being  equally  reliable.  These  cells  are  undoubtedly  more 
active  than  are  the  other  mononuclears,  the  lymphocytes,  in  ingesting 
intravascular  particulate  matter,  but  by  no  means  all  of  them  are  ever 
thus  concerned.  Furthermore,  the  oxydase  reaction  is  notoriously 
unreliable,  not  merely  differing  in  its  results  in  the  same  material 
when  applied  by  different  hands  or  in  different  ways  but  also,  as 
Menten  has  shown,  differing  in  different  animals.  The  reaction  is  often 
given  incompletely  at  best  in  the  guinea  pig  and  may  fail  in  the  case 
of  the  rabbit.  It  must  also  be  stated  against  McJunkin  that  the 
macrophages  as  a  class  do  not  respond  to  the  oxydase  reaction. 

In  spite  of  the  above  statements,  evidence  has  recently  been  accu- 
mulating to  show  that  there  is  nevertheless  some  sort  of  relation  be- 
tween the  transitionals  of  Ehrlich  and  the  class  of  great  phagocytic 
cells.  In  this  connection  work  in  clinical  haematology,  above  all  that 
of  Victor  Schilling,  has  shown  that  in  certain  conditions  transitional 
leucocytes  or  monocytes  (Naegeli)  are  specifically  increased,  giving 
a  true  monocytosis ;  in  other  conditions  the  macrophages  are  abundant 
without  notable  increase  of  the  monocytes,  giving  macrophagocytosis ; 
and  in  still  other  conditions  the  macrophage  response  is  distinctly 
associated  with  increase  of  the  monocytes.  The  latter  class  of  condi- 
tions, furnishing  an  intermediate  step  or  link  between  the  two  types 
of  response,  would  appear  to  suggest  strongly  a  biological  relationship 
in  the  two  cells. 

It  is  now  necessary  to  comment  briefly  on  the  criteria  for  recogniz- 
ing the  transitionals  or  monocytes.  Until  very  recently  the  mono- 
nuclear leucocytes  could  merely  be  classified  as  "small"  (about  the 
size  of  a  red  cell)  and  "large,"  and  in  the  latter  group  would  be 
found  tlie  forms  approaching  considerable  dimensions  and  with  con- 
cave or  horseshoe  nuclei.  We  are  now  satisfied  that  the  latter  cells  do 
not  normally  possess  neutrophilic  granulations  and  that  the  term 
"transitional"  in  Ehrlich 's  sense  is  a  complete  misnomer.  The  intro- 
duction of  the  Romanovsky  methods  of  staining  has,  however,  shown 
that  the  old  Ehrlich  large  mononuclear  and  transitional  group  does, 
in  fact,  belong  together  and  can  be  separated  from  the  lymphocytes — 
this  by  virtue  of  the  fact  that  the  transitional  group  to  which  Naegeli 
has  now  applied  the  term  "monocyte"  possesses  in  its  cytoplasm  a 
special,  fine,  dustlike  azurophilic  granulation.     Only  those  studies  on 


1921]  Simpson:  Endothelial  Macrophages  15 

the  blood  which  have  been  carried  out  by  means  of  the  best,  improved, 
modern  Giemsa  stain  can  lay  claim  to  a  justifiable  separation  of  the 
mononuclear  leucocytes  into  the  monocyte  and  lymphocyte  group. 
The  reaction  is  given  beautifully  by  the  blood  of  man.  It  is  unfortu- 
nate that  it  may  be  secured  only  with  great  difficulty,  if  at  all,  in  cer- 
tain mammalia.  The  monocytes  are  frequently  difficult  to  recognize 
in  this  way  with  dependability  in  the  rabbit.  It  is  hence  of  much 
importance  that  other  methods  for  detecting  this  group  in  the  mono- 
nuclear blood  cells  be  devised.  The  writer  has  previously  reported 
that  the  method  of  vital  staining  by  means  especially  of  Neutral  Red 
but  also  with  Brilliant  Cresyl  Blue,  Nile  Blue  Sulfate  and  a  consid- 
erable number  of  other  dyes  does  furnish  such  a  method.  It  has  hence 
seemed  of  much  importance  for  an  experimental  study  to  be  under- 
taken in  animals  in  which  the  monocytes  could  be  recognized  with 
certainty  and  in  which  at  the  same  time  the  production  of  considerable 
numbers  of  true  circulating  macrophages  could  be  brought  about.  The 
present  studies  undertaken  at  the  suggestion  of  Professor  Evans  and 
with  his  aid  aim  to  accomplish  that  task. 

Rabbits  were  submitted  to  chronic  intravenous  dosage  with  various 
materials  and  at  time  intervals  of  every  few  days  for  a  total  time 
interval  varying  from  a  month  to  four  or  five  mouths,  the  majority 
of  the  cases  being  of  the  latter  duration.  In  one  or  two  instances 
animals  were  treated  for  a  little  over  a  year.  Intravenous  injections 
were  made  of  the  colloidal  dyes  Niagara  blue  2B  and  lithium  carmine, 
of  the  larger  colloids  and  suspensoids  constituted  by  red  gold  in 
sodium  lysalbinate,  India  ink  and  lamp  black  in  gelatin  solution,  and 
with  certain  proteins  or  split  products  of  proteins,  namely,  gelatin 
and  sodium  lysalbinate.  An  obvious  point  in  common  between  the 
three  kinds  of  stimulating  agents  is  that  the  solutions  injected  always 
contained  substances  in  the  colloidal  state.  Blood  was  continually 
studied  as  obtained  in  the  living  animal  from  the  right  and  left  ven- 
tricle, and  from  the  ear  veins.  It  is  surprising  how  few  macrophages 
are  found  in  the  peripheral  blood  even  after  prolonged  stimulation  of 
the  macrophage  producing  organs.  It  is  possible  to  prove  that  this  is 
due  to  the  interposition  of  the  pulmonary  circulation,  which  filters 
out,  as  it  were,  the  great  cells,  so  that  the  latter  do  not  reach  the  peri- 
pheral blood  in  appreciable  quantities,  even  when  poured  into  the  right 
heart  in  enormous  numbers.  The  method  of  right  ventHcvMr  punc- 
ture in  the  living  animal,  though  involving  some  difficulty,  is  conse- 
quently essential  for  the  discovery  of  the  time  and  extent  of  production 


16  TJmversity  of  California  Puhlications  in  Anutomy        [Vol.  l 

of  these  cells.  In  all  cases  the  blood  was  withdrawn  quickly  with  oiled 
or  paraffined  instruments  and  submitted  to  a  supra-vital  stain  by  a 
combination  of  Janus  Green  and  Neutral  Red  as  described  previously. 
Fixed  specimens  were  stained  with  combined  Jenner-Giemsa,  Wilson's 
stain,  Graham's  oxydase,  McJunkin's  oxydase  and  combined  benzidine- 
polychrome.  At  the  time  of  autopsy  similar  studies  were  made  both 
by  the  method  of  fixed  specimens  and  by  vital  stains  of  living  films 
of  blood  from  all  the  great  veins.  As  reported  by  Kiyono,  the  blood 
in  the  splenic  and  hepatic  veins  was  always  richer  in  macrophages  than 
in  other  vessels.  High  counts  of  these  cells  in  these  veins  have  been 
paralleled  by  histological  evidence  of  the  direct  production  of  the 
macrophages  by  the  splenic  and  hepatic  endothelium,  as  has  been  shown 
by  Kiyono  and  Evans. 

The  difference  in  the  macrophage  content  of  the  blood  of  the  two 
sides  of  the  heart  in  the  living  animal  may  be  truly  spectacular  when 
the  macrophages  are  abundant,  for  enormous  numbers  may  exist  in  the 
right  and  almost  none  in  the  left  ventricle.  The  method  of  right  ven- 
tricular puncture  has  shown  that  a  hitherto  unsuspected  massive  pro- 
duction of  macrophages  may  occur  a  short  time  after  administration  of 
the  stimulating  agent  in  the  course  of  these  long  or  chronic  treatments. 
Indeed,  the  right  ventricle  has  been  observed  when  90  per  cent  of  the 
leucocytes  were  macrophages,  the  left  ventricle  containing  simul- 
taneously less  than  .1  per  cent  of  the  cells.  Our  method  of  treatment 
and  examination  of  the  blood  was  hence  ideally  adapted  to  discover 
the  peculiarities  of  these  hitherto  rare  inhabitants  of  the  blood,  and 
of  their  possible  relationships  or  transformation  from  other  blood 
cells,  becaiise  great  numbers  of  the  cells  were  at  our  disposal. 

One  of  the  chief  physiological  points  coming  out  of  the  work  has 
been  the  demonstration  that  the  macrophages  are  not  present  con- 
tinually even  in  the  venous  heart  blood  of  chronically  injected  animals. 
They  appear  in  shoivers,  i.e.,  in  chronically  treated  animals  the  time  is 
finally  reached  when  the  animal  responds  to  every  intravenous  injec- 
tion of  the  stimulating  agent  within  a  time  varyin'g  from  a  few  hours 
to  somewhat  over  a  day  by  pouring  forth  great  numbers  of  macrophages 
into  the  general  venous  circulation. 

There  is  also  an  equally  interesting  abrupt  disappearance  of  macro- 
phages from  the  circulation  and  such  a  phenomenon  may  occur  within 
a  few  minutes.  The  right  ventricle  may  show  70  or  80  per  cent  of  the 
white  cells  as  great  macrophages  and  a  second  puncture  fifteen  minutes 


i^-i]  Simpson:  E ndothelial  Macrophages  17 

later  disclose  but  1  or  2  per  cent  of  these  cells.^  In  all  cases  what  have 
been  termed  typical  macrophages  were  very  large  cells  exceeding  the 
dimensions  of  any  leucocyte,  even  the  largest  of  the  transitionals. 
Their  nuclei  were  either  round,  oval,  or  somewhat  indented  and  a  few 
instances  were  encountered  of  cells  containing  two  or  even  three  nuclei. 
Furthermore,  mitotic  figures  were  occasionally  observed.  The  cyto- 
plasm usually  contains  granules  and  vacuoles  in  variable  quantities 
often  arranged  radially  around  the  centriolar  apparatus,  and  fre- 
quently contains  phagocytized  material  in  the  form  of  either  frag- 
mented red  or  white  cells,  as  well  as  some  of  the  stimulating  substances 
which  had  been  injected  intravenously.  With  Giemsa  stains  the  cyto- 
plasm is  packed  with  fine  azurophilic  granulations.  Except  for  the 
phagocytized  material  or  foci  the  cytoplasm  was  negative  in  its  re- 
sponse to  oxydase  tests.  The  cells  are  beautiful  when  studied  with 
supra-vital  stains.  With  Brilliant  Cresyl  Blue  the  segregation  appa- 
ratus is  violet  and  variable  in  amount,  the  phagocytized  material  stain- 
ing very  early  and  intensely.  The  mitochondria  are  unstained.  The 
refractive  vacuoles  and  certain  non-refractive  vacuoles  are  unstained. 
With  Neutral  Red  and  Janus  Green  the  segregation  apparatus  and 
phagocytized  material  varies  from  orange  to  red,  while  the  mito- 
chondria are  blue  green.  It  is  remarkable  that  the  macrophages  do 
not  contain  the  injected  material  in  appreciable  quantities  during  the 
greater  part  of  the  time  in  which  they  are  poured  into  the  blood 
stream.     This  indicates  a  great  over-production  of  the  cells. 

Some  other  significant  changes  in  the  blood  seem  associated  with 
the  production  of  circulating  macrophages.  During  the  time  of  the 
showers  when  the  right  ventricular  content  of  macrophages  was  high, 
the  blood  from  this  chamber  of  the  heart  would  be  thick  and  stringy, 
contrasting  with  the  thin  arterial  blood  of  the  left  ventricle.  The 
clotting  time  of  this  viscuous  blod  may  also  be  greatly  prolonged  or  a 


lA  striking  clinical  illustration  of  the  same  "shower  phenomenon"  was 
fortunately  obtained  in  the  medical  service  of  the  University  of  California 
Hospital  during  ther  months  of  May  and  June,  1921.  These  were  the  two  termi- 
nal months  of  the  course  of  a  fatal  case  of  Libman  's  subacute  bacterial  endo- 
carditis occurring  in  a  young  man  eighteen  years  of  age.  I  am  indebted  to 
Doctors  Moifitt,  Kerr,  and  Sampson  for  permission  to  observe  and  for  much  aid 
in  the  study  of  this  interesting  case. 

Positive  blood  cultures  were  obtained  of  non-haemolytic  streptococcus  viri- 
dans.  Macrophages  were  found  varying  from  1  to  1%  per  cent  in  the  ear  vein 
blood  at  various  times  and  on  one  occasion  (June  11)  14  per  cent  of  these  great 
cells  were  encountered,  but  on  the  same  day,  four  hours  later,  less  than  1  per 
cent  of  the  cells  were  present.  There  were  occasions  when  none  of  the  cells 
could  be  detected  in  the  blood  and  in  all  respects  the  spasmodic  or  showerlike 
behavior  of  the  macrophages  seen  in  experimental  animals  was  encountered  here 
in  man. 


18  Umversity  of  Calif arnia  Puhlications  in  Anatomy        \y^^- 1 

clot  indeed  fail  to  form  when  similar  simultaneous  left  ventricular 
samples  clot  normally.  Furthermore,  the  peculiar  right  ventricular 
blood  has  a  reduction  or  practical  disappearance  of  platelets.  In  all 
cases  differential  white  counts  were  made  by  examining  at  least  two 
hundred  cells  in  the  living  supra-vitally  stained  films.  In  this  way 
it  was  possible  to  gain  an  accurate  idea  of  the  behavior  of  that  interest- 
ing group  of  normal  mononuclear  blood  cells  whose  relationship  to  the 
macrophages  was  sought,  namely,  the  monocytes. 

The  supra-vital  method  shows  a  striking  parallelism  in  the  behavior 
of  macrophages  and  monocytes,  a  parallelism  not  shown  by  the  lym- 
phocytic cells.  But  of  greater  importance  is  the  fact  that  during  the 
time  of  occurrence  of  the  so-called  macrophage  showers,  all  inter- 
mediate cell  types  are  encountered,  bridging  the  gap  between  norm<il 
monocytes  and  macrophages.  It  is  furthermore  of  interest  that,  with 
many  animals,  shortly  preceding  macrophage  showers  the  monocytes 
were  observed  to  increase  numerically,  and  during  the  shower  were  to 
some  extent  filtered  out  by  the  lungs  just  as  the  macrophages  are  almost 
completely  filtered  out  by  the  pulmonary  circulation.  This  evidence 
would  appear  at  any  rate  to  now  point  convincingly  to  a  kinship 
between  the  two  kinds  of  cells. 

Inasmuch  as  the  monocytes,  however,  are  not  effectively  filtered 
and  circulate  generally,  a  very  rapid  production  of  the  monocytes  in 
any  region  would  be  necessary  to  appreciably  raise  the  monocyte  count 
of  blood  from  those  regions  and  hence  throw  light  in  this  way  on.  the 
sites  of  origin  of  these  cells.  Such  significant  differences  were  not 
observed. 

These  studies  appear  to  give  the  first  certain  indication  of  the 
relationship  between  the  monocyte  and  macrophage,  and  yet  if  the  same 
mother  endothelium  is  supposed  to  proliferate  these  two  types  of  cell, 
we  must  regard  macrophage  production  as  a  pathological  or  atypical 
response.  Certain  it  is  that  the  macrophages  can  not  usually  be  re- 
garded as  the  result  merely  of  further  growth  of  the  monocyte  cell, 
for  there  would  then  probably  appear  far  greater  numbers  of  these 
only  slightly  hypertrophied  monocytes  in  the  general  circulation. 
This  is  never  the  case.  During  the  prolonged  treatments  of  our  animals 
and  between  the  showers  of  macrophages,  the  monocytes  always  con- 
stitute a  distinct  cell  group  Avith  not  conspicuously  larger  members 
even  though  their  numbers  may  be  increased.  It  does  not  appear  now 
so  remarkable  that  macrophages  are  not  more  frequently  encountered 
in  many  conditions  since  we  know  of  the  filtration  by  the   lungs. 


1921]  Simpson:  Endothelial  Macrophages  19 

Furthermore,  the  splenic  blood  from  several  normal  animals  has  been 
examined  and  seems  to  always  contain  at  least  a  few  true  macrophage 
cells.  It  is  important  for  us  to  realize  that  in  addition  to  these  facts 
which  indicate  that  macrophages  are  not  merely  transformed  mono- 
cytes, there  are  other  facts  which  indicate  certain  differences,  as 
well  as  the  affinities  on  which  we  have  already  dwelt,  between  these 
two  groups  of  cells.  Mention  here  must  be  made  of  the  fact  that  the 
monocytes,  contrary  to  the  opinion  of  Aschoff  and  Kiyono,  in  animals 
treated  chronically  with  any  of  these  agents,  do  not  contain  the  agent 
in  question.  They  are  not  stained  vitally  by  the  benzidine  or  carmine 
dye,  and,  while  macrophages  are  sometimes  not  so  stained,  the  reintro- 
duction  of  the  vital  dye  into  the  blood  stream  of  the  living  animal 
will  result  in  the  staining  of  the  macrophages  but  not  of  the  monocytes. 
While  the  present  work,  then,  may  be  said  to  point  strongly  to  the 
biological  affinities  of  these  two  interesting  cell  types,  it  cannot  be 
said  to  show  an  identity  or  that  one  may  be  transformed  into  the 
other  in  the  free  or  circulating  blood  stream.  This  evidence  is  in 
consonance  with  all  that  we  know  about  a  similar  lack  of  transforma- 
tion of  one  type  of  leucocyte  to  another  in  the  blood  current,  our 
information  tending  to  bring  the  conviction  that  a  cell  once  shed  into 
the  stream  thenceforth  usually  undergoes  no  significant  transformation. 

Transmitted  September  6,  1921. 
(See  footnote,  page  1.) 


<( 


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